Pertussis toxin exerts its effects on mammalian cells by ADP-ribosylating regulatory proteins involved in signal transduction. The toxin comprises two components, an enzymatically active A subunit and a B oligomer needed for binding to the eukaryotic cell. ATP plays an important role in the regulation of pertussis toxin activity. Upon entry of the toxin into the eukaryotic cell, ATP is thought to bind to the toxin, promote disruption of the toxin subunits, thus activating the enzymatic activity of the toxin molecule. We determined that the binding site for ATP is located on the B oligomer. Binding of ATP to both the holotoxin and B oligomer was studied in detail in the hope of better understanding regulation of pertussis toxin activity. Additional studies have demonstrated that a pertussis toxin molecule can be assembled in vitro from the native B oligomer and a recombinant A subunit which had an altered amino acid and thus reduced enzymatic activity. This species exhibited markedly lower biological activity as measured by the CHO cell assay. These studies indicate the pertussis toxin assembled from polypeptides derived from overexpression in recombinant systems may be considered as a component of new, safer acellular pertussis vaccines. Currently, studies have been initiated to examine the mechanism of entry of pertussis toxin into eucaryotic cells. Several bacterial toxins including diphtheria toxin enter cells via receptor-mediated endocytosis. Exposure of these toxins to the acidic environment of the endosome causes conformational changes in the protein resulting in entry of the toxin into the cell cytosol. We have examined whether pertussis toxin might also require an acidic environment for entry into the eucaryotic cell. Neither NH4Cl nor monensin affect pertussis toxin action but both agents inhibit diphtheria toxin entry into the cell suggesting that pertussis toxin does not require an acidic environment for entry.